Leishmania parasites cause a spectrum of devastating diseases in humans known as leishmaniasis. Current drug treatments are inadequate, often toxic, and no safe vaccine is available. A major obstacle in the development of new therapies is the lack of understanding on how Leishmania parasites evade immune activation and persist in mammalian hosts. The long-term goal is to elucidate the roles of parasite lipid metabolism in Leishmania virulence. In comparison to membrane proteins and glycoconjugates, the biology of lipids has been extremely understudied in protozoan pathogens. The objective of this application is to determine the molecular mechanism by which the turnover of inositol phosphorylceramide (IPC) contributes to the infectivity of Leishmania parasites. IPC is a parasite-specific lipid and preliminary studies suggest its degradation (catalyzed by a putative inositol phosphorylceramidespecific phospholipase C or IPC-PLC) is essential for Leishmania virulence. The central hypothesis is that IPC-PLC plays indispensable roles in the subversion of host immune defense and the survival of amastigotes in mammals. The rationale for this project is that such new knowledge may lead to the identification of parasite-specific targets for selective chemotherapy against Leishmania. Guided by strong preliminary data, the central hypothesis will be tested by pursuing four specific aims: 1) evaluate the impact of IPC-PLC on the expression of virulent traits in promastigotes;2) determine the molecular interaction between IPC-PLC null promastigotes and mammalian hosts;3) evaluate the importance of stage- and organelle-specific expression of IPC-PLC in Leishmania virulence;and 4) purify IPC-PLC and determine its biochemical properties. Under the first and second aims, a null mutant of IPC-PLC (already in hand) will be used to unmask the contribution of IPC degradation to the establishment and persistence of Leishmania infection in macrophages and mice. Under the third aim, the activity of IPC-PLC will be introduced into the null mutant under strict spatial or temporal control;effects of such stage- and organelle-specific expression of IPC-PLC on virulence will be evaluated in promastigotes and amastigotes. Under the final aim, IPC-PLC will be purified either as a recombinant protein (from E. coli) or as an endogenous protein (from Leishmania parasites) and its biochemical properties will be determined. The proposed research is innovative and highly significant, because it is expected to identify, for the first time, a crucial link between the degradation of an abundant membrane lipid (IPC) and virulence in Leishmania parasites. In addition, because IPC and IPC-PLC are not present in humans, such findings are expected to identify and validate new targets for selective therapy to control these dangerous pathogens.